The dystrophin-deficient muscle diseases include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and X-linked dilated cardiomyopathy (XLDC). They are among the most common genetic diseases and affect more than one male infant in every 3,000 newborns. The progressive muscle degeneration and weakness usually confine the patients to wheelchairs by their early teens, and lead to death by their early twenties. Women carriers are also affected in their 40s to 50s. The above mentioned muscle diseases are caused by mutations in the dystrophin gene, specifically, the X-linked recessive mutations. The dystrophin gene is the largest gene known to date, which spans nearly 2.4 million base-pairs on the X-chromosome with 79 exons, a coding sequence of about 11.5 kb, and a high rate of de novo mutations.
Currently, there is no effective treatment for dystrophin-deficient diseases. Novel genetic approaches including cell therapy and gene therapy have been actively explored. However, the tremendous size of the dystrophin gene and mRNA (14 kilobases; kb) are formidable obstacles to the development of gene therapy. For this reason, studies have focused on developing smaller abbreviated versions of the mini-dystrophin or micro-dystrophin genes. These abbreviated genes can be introduced via a viral vector (such as an AAV vector and a lentiviral vector) or via stem cells (such as mesoangioblasts, CD133+ stem cell, and side population cells) (Yuasa et al., 1998; Wang et al., 2000; Ferrer et al., 2000; Harper et al., 2002; Fabb et al., 2002; Sakamoto et al., 2002; Bachrach et al., 2004; Sampaolesi et al., 2006; Benchaouir et al 2007, Cell Stem Cell 1:646-657). The microgene generally refers to the naturally occurring and the synthetic dystrophin genes that have a coding sequence equal to or less than 5 kb and can be packaged in a single adeno-associated viral vector (AAV). The minigene refers to the synthetic dystrophin genes that have a coding sequence equal to or less than 10 kb, but larger than 5 kb. The minigene cannot be packaged in a single AAV vector but can be delivered through a variant of dual vectors such as the overlapping vector, the trans-splicing vector and the hybrid vector.
The wild type dystrophin gene carries two main biological functions. One is to provide a mechanic link between the cytoskeleton and the extracellular matrix so that the muscle membrane is stabilized during contraction. The other is to provide signaling function for a number of important cellular activities. The signaling function of dystrophin is accomplished mainly through a partner protein called neuronal nitric oxide synthase (nNOS) (Rando, 2001). Several studies have shown that nNOS is recruited to the sarcolemma by the full-length dystrophin protein (Brenman et al., 1995; Brenman et al., 1996). Recent studies further suggested that the loss of nNOS in dystrophin-deficient muscle contributed significantly to the disease progression in animal models of DMD and human DMD patients (Brenman et al., 1995; Chang et al., 1996; Thomas et al., 1998; Sander et al., 2000). Furthermore, transgenic over-expression of nNOS ameliorates muscle pathology in the mdx mouse model of DMD (Wehling et al., 2001; Tidball and Wehling-Henricks, 2004; Shiao et al., 2004; Wehling-Henricks et al., 2005).
Attempts to generate dystrophin minigenes and microgenes have been documented. For example, the ΔR4-R23/ΔC micro-dystrophin can reduce histopathology in mouse models of DMD. However, this micro-protein cannot recover muscle specific force to the normal level (Harper et al., 2002; Gregorevic et al., 2004; Liu et al., 2005; Yue et al., 2006; Gregorevic et al., 2006). The ΔH2-R19 mini-dystrophin gene is derived from a very mild patient (England et al., 1990; Harper et al., 2002). This minigene is better than the ΔR4-R23 microgene or the ΔR4-R23/ΔC microgene because it can recover the muscle specific force to the same level as the full-length dystrophin gene (Harper et al., 2002; Lai et al., 2005). However, the minigene cannot restore nNOS. As a matter of fact, none of the existing mini- or micro-dystrophin genes have the ability to recruit nNOS to the sarcolemma (Table 1) (Chao et al., 1996; Crawford et al., 2000; Warner et al., 2002; Wells et al., 2003; Torelli et al., 2004; Lai et al., 2005; Yue et al., 2006; Li et al., 2006; Judge et al., 2006). The failure to restore sarcolemmal nNOS will significantly reduce the therapeutic efficacy of the minimized dystrophin genes.
Previously it was thought that nNOS is recruited to the sarcolemma through the C-terminal domain of the dystrophin protein (Brenman et al., 1995; Brenman et al., 1996). The full-length dystrophin protein has four domains including the N-terminal domain, mid-rod domain, cysteine-rich domain, and C-terminal domain. The N-terminal domain and a portion of the mid-rod domain interact with cytoskeleton protein F-actin. The mid-rod domain contains 24 spectrin-like repeats and four hinges. The cysteine-rich domain interacts with transmembrane protein dystroglycan to connect dystrophin to the extracellular matrix. The C-terminal domain contains two syntrophin binding sites and one dystrobrevin binding site. Several studies suggest that nNOS is recruited to the sarcolemma through a PDZ/PDZ domain interaction between nNOS and α-syntrophin (Brenman et al., 1996; Hillier et al., 1999; Kameya et al., 1999; Tochio et al., 1999; Adams et al., 2001; Miyagoe-Suzuki and Takeda, 2001). This seems to suggest that a mini- or micro-gene with the C-terminal domain should be able to recruit nNOS to the sarcolemma. However, studies have repeatedly shown that none of the existing C-terminal domain containing mini/micro-dystrophin proteins can bring nNOS to the sarcolemma although syntrophin is perfectly localized to the sarcolemma in these occasions (Chao et al., 1996; Lai et al., 2005). Furthermore, a C-terminal truncated full-length dystrophin protein was shown to recruit nNOS to the sarcolemma (Crawford et al., 2000).
Table 1 evaluates and summarizes the structures and functions of several naturally occurring dystrophin genes and several representative synthetic dystrophin minigenes and microgenes. Among all the tested genes, only the natural isoform Dp427 (full-length dystrophin gene) and Dp260 (retinal isoform of the full-length gene), and a C-terminal truncated full-length gene (Δ71-78) have been confirmed to restore nNOS to the sarcolemma. However, as discussed above, these genes are too big for AAV or lentiviral vector packaging.
Therefore, there is a need for developing a novel mini/micro-dystrophin gene, or a series thereof, with the ability to restore nNOS to the sarcolemma, and to be used for gene therapy in the treatment of DMD, BMD and XLDC.